Circulating HMGB1 Levels Are Associated With Glucose Clamp-Derived Measures of Insulin Resistance in Women With PCOS

Context: High mobility group box 1 (HMGB1) is a nuclear constitutive and highly conserved mammalian protein which shows several important physiologic functions, playing a crucial role in local and systemic inflammation. It also seems to be implicated in the development of metabolic syndrome, whereas some in vitro and animal models and recent preliminary human studies suggested its potential role in polycystic ovary syndrome (PCOS) pathophysiology. In particular, some data suggested a potential role of HMGB1 in follicular maturation block. Moreover, increased blood and follicular fluid HMGB1 concentrations have been reported in PCOS women, showing a direct correlation with surrogate indexes of insulin-resistance (IR) (i.e. HOMA-IR). Objective: The purpose of the present study was to investigate the relationships between serum HMGB1 levels and clinical, endocrine and metabolic parameters of PCOS patients. Design and patients: Sixty women with PCOS, 30 with IR and 30 with normal insulin sensitivity (IS), and 30 healthy controls were included in the study. In these subjects, body fat was quantified by bioelectrical impedance; plasma HMGB1 levels were measured using a specific ELISA method (Tecan, Mannedorf, Switzerland); and serum androgens were measured by liquid chromatography/mass spectrometry and equilibrium dialysis. In PCOS women, IR was measured using the gold standard hyperinsulinemic-euglycemic clamp technique, combined with indirect calorimetry. Results: HMGB1 levels did not differ between PCOS women and healthy controls (4.11 ± 3.22 vs 3.77 ± 2.50 ng/mL, respectively; p=0.61). Moreover, HMGB1 levels did not differ between PCOS phenotype subgroups. However, PCOS IR women showed higher levels of this protein as compared with PCOS IS (5.00 ± 3.53 vs 3.16 ± 2.59 ng/mL, respectively; p=0.017). In women with PCOS, HMGB1 levels were associated with several metabolic parameters, including IR measured by glucose utilization during the clamp (rho -0.37, p=0.005). This correlation was preserved after adjusting for potential confounding parameters, such as age, fat mass and serum free testosterone. HMGB1 levels did not change during glucose-clamp induced acute hyperinsulinemia, either in the whole cohort of patients or in IR and IS subgroups analyzed separately. Both in the whole population under study and in PCOS women, HMGB1 levels did not correlate with anthropometric parameters, hormonal features and ovarian morphology. Conclusions: In women with PCOS, HMGB1 blood levels show an independent association with insulin resistance. However, no associations with other typical features of the syndrome were found. Further research is needed in order to establish whether this protein may play a role in the pathogenesis of PCOS.

Cold-induced dishabituation in rodents exposed to recurrent hypoglycaemia

Aims/hypothesis Recurrent hypoglycaemia in people with diabetes leads to progressive suppression of counterregulatory hormonal responses to subsequent hypoglycaemia. Recently it has been proposed that the mechanism underpinning this is a form of adaptive memory referred to as habituation. To test this hypothesis, we use two different durations of cold exposure to examine whether rodents exposed to recurrent hypoglycaemia exhibit two characteristic features of habituation, namely stimulus generalisation and dishabituation. Methods In the first study (stimulus generalisation study), hyperinsulinaemic–hypoglycaemic (2.8 mmol/l) glucose clamps were performed in non-diabetic rodents exposed to prior moderate-duration cold (4°C for 3 h) or control conditions. In the second study (dishabituation study), rodents exposed to prior recurrent hypoglycaemia or saline (154 mmol/l NaCl) injections over 4 weeks underwent a longer-duration cold (4°C for 4.5 h) exposure followed 24 h later by a hyperinsulinaemic–hypoglycaemic (2.8 mmol/l) glucose clamp. Output measures were counterregulatory hormone responses during experimental hypoglycaemia. Results Moderate-duration cold exposure blunted the adrenaline (epinephrine) response (15,266 ± 1920 vs 7981 ± 1258 pmol/l, Control vs Cold; p < 0.05) to next day hypoglycaemia in healthy non-diabetic rodents. In contrast, the suppressed adrenaline response (Control 5912 ± 1417 vs recurrent hypoglycaemia 1836 ± 736 pmol/l; p < 0.05) that is associated with recurrent hypoglycaemia was restored following longer-duration cold exposure (recurrent hypoglycaemia + Cold 4756 ± 826 pmol/l; not significant vs Control). Conclusions/interpretation Non-diabetic rodents exhibit two cardinal features of habituation, namely stimulus generalisation and dishabituation. These findings provide further support for the hypothesis that suppressed counterregulatory responses following exposure to recurrent hypoglycaemia in diabetes result from habituation. Graphical abstract

Precision and Accuracy of Hyperglycemic Clamps in a Multicenter Study

Background: Application of glucose clamp methodologies in multicenter studies brings challenges for standardization. The Restoring Insulin Secretion (RISE) Consortium implemented a hyperglycemic clamp protocol across seven centers using a combination of technical and management approaches to achieve standardization. Methods: Two- stage hyperglycemic clamps with glucose targets of 200 mg/dL and >450 mg/dL were performed utilizing a centralized spreadsheet-based algorithm that guided dextrose infusion rates using bedside plasma glucose measurements. Clamp operators received initial and repeated training with ongoing feedback based on surveillance of clamp performance. The precision and accuracy of the achieved stage-specific glucose targets were evaluated, including differences by study center. We also evaluated robustness of the method to baseline physiologic differences and on-study treatment effects. Results: The RISE approach produced high overall precision (3-9% variance in achieved plasma glucose from target at various times across the procedure) and accuracy (SD <10% overall). Statistically significant but numerically small differences in achieved target glucose concentrations were observed across study centers, within the magnitude of the observed technical variability. Variation of the achieved target glucose over time in placebo-treated individuals was low (<3% variation), and the method was robust to differences in baseline physiology (youth vs adult, IGT vs diabetes status) and to differences in physiology induced by study treatments. Conclusions: The RISE approach to standardization of the hyperglycemic clamp methodology across multiple study centers produced technically excellent standardization of achieved glucose concentrations. This approach provides a reliable method for implementing glucose clamp methodology across multiple study centers.

The Effects of Self-Control on Glucose Utilization in a Hyperinsulinemic Euglycemic Glucose Clamp

Background. The glucose hypothesis of self-control posits that acts of self-control may draw upon glucose as a source of energy, leading to a decrease in blood glucose levels after exerting self-control, mirroring the temporary depletion of self-control, but supporting evidence is mixed and inconclusive. This might partly be due to using methods that are not suitable to reliably quantify glucose utilization. Aims. We aimed at examining whether self-control exertion leads to an increase in glucose utilization. Method. In a sample of N = 30 healthy participants (50% women, age 26.5 ± 3.5 years) we combined a hyperinsulinemic euglycemic glucose clamp (a well-established and validated procedure in experimental endocrinology to reliably quantify systemic glucose utilization) with a standard self-control dual-task paradigm. In the first task, the experimental group completed a variation of a paper-and-pencil crossing out letter task (COLT) that demanded self-control; the control group completed a variation of the COLT that did not demand self-control. The second task for both groups was a computerized two-color word Stroop which demanded self-control. Results. We did not find a significant main effect for time, nor a time × group interaction with respect to glucose utilization, which indicates that glucose utilization did not differ significantly over time or between groups. Limitations. Due to the time-consuming and complicated clamp method, our sample was rather small. Conclusion. Our results revealed little evidence for the notion that self-control efforts are associated with a relevant increase in peripheral glucose utilization.